MRSA and VRE strains kill C. elegans using traits that contribute to the pathogenesis of infection in mammals, and this model system can be used to study host-pathogen interactions involving staphylococcal and enterococcal virulence factors. Importantly, Staphylococcus aureus virulence determinants involved in mammalian pathogenesis, including the quorum-sensing global virulence regulatory system agr, the global virulence regulator sarA, and the alternative sigma factor sigma(B) are required for full pathogenicity in nematodes. Based on these observations, we developed a C. elegans-S. aureus screening assay that is performed using the 384-well plate format. This whole animal assay can be used to screen compound libraries, allowing the identification of compounds that prevent host killing that would not be detected in traditional in vitro screens. Our objective is to implement this C. elegans-based assay and utilize automated, high-throughput, whole animal screens to identify compounds with efficacy against MRSA, VRE and VRSA. Identified compounds will be evaluated by carrying out dose-response and time-course studies, determining in vitro MICs, determining toxicity, and by prioritizing the compounds for testing in mammals. We also propose to characterize the compounds previously identified in a C. elegans - E. faecalis screen, and synergize with other Subprojects in order to study compounds identified through the C. elegans assays and characterize compounds from other Subrojects. The C. elegans-based assays are particularly appealing in that they allow concurrent evaluation of toxicity and antimicrobial activity, as well as study of bacterial cells that are in a non-planktonic form. Thus false leads can be eliminated from further consideration early, and new leads will be clearly identified. In addition to compounds that have direct antibacterial activity, the C. elegans-based assays will identify compounds that affect bacterial virulence factors and host function, providing new leads, but also new chemical biology tools for exploring host/pathogen interaction.